Application of gordonibacter pamelae in animal feed and preparation process of oral preparation thereof

Abstract

The application belongs to the field of biotechnology feed animal breeding, and particularly relates to application of Gordonia alkanivorans in animal feed and preparation process of oral preparation of the Gordonia alkanivorans. The process comprises processes such as slant culture, shake flask culture, seed culture and fermentation culture to prepare a liquid cell solution or processes such as membrane separation, low-temperature high-speed centrifugation, granulation and vacuum low-temperature drying to prepare a solid granular preparation. The preparation is rich in carotenoids (lycopene), peptidoglycan, amino acids, proteins and flavonoid nutritional ingredients. The Gordonia alkanivorans Weicao-1 has the advantages of promoting animal growth, enhancing the disease resistance of animals, improving the production performance of animals and improving meat quality. The application range is wide, and the application is related to live pigs and aquatic products, and is convenient for popularization.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology feed animal breeding, specifically the application of alkali-eating Gordon's bacterium in animal feed and its oral preparation process. Background Technology

[0002] The discovery of antibiotics has had a profound impact on human production and life, and feed antibiotics have been widely used. Besides significantly improving livestock and poultry productivity, they have also played a crucial role in preventing and resisting diseases and promoting growth. However, over time, people have gradually discovered that long-term use of antibiotics has many drawbacks, such as drug residues in livestock, poultry, and egg products.

[0003] As people's living standards continue to improve, their demands for the quality of livestock, poultry, and egg products are becoming increasingly urgent. To safeguard public health, my country's Ministry of Agriculture and Rural Affairs issued Announcement No. 194 in July 2019, requiring the withdrawal of all growth-promoting drug feed additives, except for traditional Chinese medicine, by the end of 2020. my country is entering an era of "antibiotic-free feed," bringing tremendous commercial opportunities for the promotion of microbial feed.

[0004] Microorganisms, as the largest group of species on Earth, possess abundant species resources. Humans have used microorganisms and their metabolites to treat diseases for thousands of years. Microecological preparations are biological agents or live bacterial preparations containing live bacteria, made from beneficial microorganisms through processes such as cultivation, fermentation, drying, and processing, and used on animals. Under modern livestock production conditions, the effective use of microecological preparations to regulate the intestinal microecological balance of livestock and poultry, enhance nutrient absorption, strengthen immunity to promote healthy growth, improve production performance, and enhance product quality has become a major trend in the feed additive industry.

[0005] The number of fungi listed in the feed catalog of my country's Ministry of Agriculture and Rural Affairs is very limited, mainly including six categories: lactic acid bacteria, yeast, Bacillus, Propionibacterium, photosynthetic bacteria, and Aspergillus. *Gordonia* is a rare actinomycete, and *Gordonia alkanivorans* is even rarer. Current patent information indicates a lack of application technology for this species in animal feed. Summary of the Invention

[0006] The purpose of this invention is to provide an application of *Gordonella alkali-eating* in animal feed and a process for preparing its oral formulation.

[0007] The objective of this invention is achieved through the following means:

[0008] A species of alkali-eating Gordon's bacterium, namely Gordonia alkanivorans weicao-1, with the accession number CGMCC No. 26361.

[0009] As a further limitation, the application of Gordonia alkanivorans weicao-1 in animal feed can effectively promote animal growth, enhance animal disease resistance, improve animal production performance, and improve meat quality.

[0010] A process for preparing an oral formulation of *Gordonella alkali-eating*, comprising the following steps:

[0011] A. Fermentation:

[0012] a. Slant seed culture: Inoculate the seed of *Gordonia alkanivorans* weicao-1 onto a slant culture medium and incubate at 30-31℃ for 24-36 hours. The culture turns red, and microscopic examination reveals no contaminants and vigorous growth.

[0013] b. Shaking flask seed culture: Inoculate the slant seed of *Gordonia alkanivorans* weicao-1 into an Erlenmeyer flask at 30-31℃, with a light intensity of 750-850 lx and a shaking speed of 150 rpm for 48 hours. The culture will turn red, and microscopic examination will show no contamination and the bacteria will be vigorous. Transfer the culture to an inoculation bottle.

[0014] c. Seed culture: Transfer the seeds from the inoculation bottle to the seed tank for culture. The initial pH of the culture medium is 6.8-7.0, the light intensity is 850-1000 lx, the aeration rate is 1:0.85-0.9, the antifoaming agent is added automatically, and the temperature is 30-32℃. After culturing for 24-26 hours, the cells turn red and samples are taken for microscopic examination. The cells are uniformly grown and free of contaminants.

[0015] d. Fermentation tank cultivation: Inoculate the seed culture from the seed tank into the fermenter at an inoculum rate of 4-8%. Maintain a temperature of 30-32℃, a light intensity of 1000-1500 lx, an aeration rate of 1:1, and automatically add antifoaming agent. Cultivate for 48-52 hours until the culture turns red to orange-red. Microscopic examination shows that the cells have aged into short rod-shaped structures, and the biomass reaches (1.0-1.5) × 10⁻⁶. 10 Fermentation was stopped when the cfu / ml concentration was reached. The carotenoid content (calculated as lycopene) in the whole-cell solution was required to be no less than 350 μg / ml to obtain the Gordonia alkanivorans Weicao-1 liquid preparation.

[0016] B. Formulate into oral solid dosage forms:

[0017] e. Membrane separation: Using an organic membrane device, the liquid preparation of *Gordonia alkanivorans* Weicao-1 obtained in step A was subjected to membrane separation and concentrated to one-tenth of its original volume;

[0018] f. Refrigerated high-speed centrifugation: Place the above concentrated solution in a refrigerated high-speed centrifuge, centrifuge at 5500-7500 rpm, control the temperature below 8℃, and centrifuge for 10-15 minutes; separate the solid and liquid, and reserve the bacterial sludge and supernatant;

[0019] g. Granulation preparation: Weigh the sludge, add 4 times the amount of skim milk powder, mix well, granulate through a 24-mesh sieve, and bake at below 60℃ and under a vacuum of -0.08 MPa for 15-20 minutes;

[0020] h. Granulation of the clear liquid: Concentrate the clear liquid to one-tenth of its volume, weigh it, add 3 times the amount of maltodextrin to refine the soft material, granulate it through a 24-mesh sieve, and bake it at 70-75℃ under a vacuum of -0.07-0.08 MPa for 2-3 hours;

[0021] i. Mixing: Mix the particles from steps g and h above to obtain a biomass of (1.5–2.5) × 10⁻⁶. 10 cfu / g yielded Gordonia alkanivorans Weicao-1 solid formulation.

[0022] As a further limitation, the culture medium in steps c and d shall be prepared according to the following formula by weight / volume percentage:

[0023] .

[0024] Gordonia alkanivorans (Weicao-1) oral preparation is an oral live bacterial preparation with high biomass and high carotenoid content. The whole-cell solution contains no less than 350 μg / mL of carotenoids (calculated as lycopene) (oral liquid preparation), which is 14 times the lycopene content in tomatoes. It also contains peptidoglycans, amino acids, proteins, flavonoids, and vitamins. Studies have shown that carotenoids and fat-soluble vitamins (C&FSV) contain long chains of isoprene (or isoprene + cyclic structure) or cyclopentane polyhydrophenanthrene structures, and their octanol-water partition coefficient is > 8, belonging to highly lipophilic food components. Therefore, they have a similar intestinal absorption mechanism to C&FSV. Studies have shown that lycopene can directly bind to free radicals, interrupting their chain reaction; it can also activate the Nrf2 antioxidant signaling pathway, thereby increasing the mRNA expression level and activity of related antioxidant enzymes; simultaneously, it reduces the activity of oxidases, thereby reducing free radical generation and accelerating free radical action, ultimately maintaining the oxidative balance of cells or the body; lycopene can weaken lipid synthesis through multiple pathways, while accelerating lipid transport and mobilization, thus achieving a lipid-lowering effect; lycopene has good anti-inflammatory and immunomodulatory activities, on the one hand by regulating the production and release of inflammatory factors; on the other hand by promoting the production of B and T lymphocytes, stimulating the normal differentiation of T cells, and enhancing the activity of natural killer (NK) cells, thereby enhancing immunity.

[0025] The production process and preparation method of *Gordonia alkanivorans* Weicao-1 are easy for industrial production and quality control. The fermentation process is further optimized, and the energy consumption of the formulation process is much lower than that of the freeze-drying process.

[0026] Gordonia alkanivorans (Weicao-1) promotes animal growth, enhances disease resistance, and improves animal production performance. It has a wide range of applications, including pigs and aquaculture, making it easy to promote.

[0027] The application of *Gordonia alkanivorans* Weicao-1 in pigs showed the following results:

[0028] (1) Significantly reduces the feed conversion ratio of weaned piglets.

[0029] (2) It significantly improved the ability of weaned piglets to scavenge free radicals, inhibited lipid peroxidation, and enhanced the body's antioxidant capacity.

[0030] (3) It significantly increased the levels of NO, IL-2, IL-12 and TNF-a in the serum of weaned piglets, promoted the secretion of SIgA in the intestinal mucosa, and improved the self-immunity of weaned piglets.

[0031] (4) It significantly inhibits the proliferation of Escherichia coli in the intestine and reduces the relative abundance of pathogenic Escherichia coli; it promotes the growth of Lactobacillus and increases the relative abundance of probiotic Lactobacillus. It maintains the balance of the intestinal flora.

[0032] (5) It can increase the height of intestinal villi and decrease the depth of crypts in weaned piglets, thereby improving the small intestine's ability to digest and absorb nutrients and thus improving production performance.

[0033] (6) By regulating the activity of gastrointestinal enzymes, the weight gain of fattening pigs can be significantly improved and the feed conversion ratio can be reduced.

[0034] (7) Significantly increases the activity of GSH-PX and SOD enzymes in the serum and tissues of fattening pigs, reduces the content of MDA, decreases the degree of oxidation in muscle, and thus improves meat quality.

[0035] The application of *Gordonia alkanivorans* Weicao-1 in shrimp showed the following results:

[0036] (1) Adding 0.05% Gordonia alkanivorans Weicao-1 preparation to the diet resulted in better growth of Litopenaeus vannamei;

[0037] (2) Adding 0.04%-0.05% of Gordonia alkanivorans Weicao-1 preparation to the diet can improve the digestive capacity of shrimp;

[0038] (3) Adding 0.03% Gordonia alkanivorans Weicao-1 preparation resulted in better intestinal villus height, width and intestinal wall muscle layer thickness in shrimp compared to other groups, which has a promoting effect on intestinal health.

[0039] (4) Adding 0.04%-0.05% of Gordonia alkanivorans Weicao-1 preparation to the diet can significantly improve the antioxidant capacity of shrimp hepatopancreas, and adding 0.05% of Gordonia alkanivorans Weicao-1 preparation can improve the immune capacity of shrimp hepatopancreas;

[0040] (5) In terms of inflammatory response, treatment with Gordonia alkanivorans weicao-1 generally up- and down-regulates the expression of pro-inflammatory factors and exerts an anti-inflammatory effect. Attached Figure Description

[0041] The present invention will now be described in further detail with reference to the accompanying drawings:

[0042] Figure 1 Colony morphology of *Gordonella alkali-eating* in a petri dish;

[0043] Figure 2 Schematic diagram of the liquid culture state of *Gordonella alkali-eating*;

[0044] Figure 3 Photograph of Gram staining of *Gordonella alkali-eating* strain;

[0045] Figure 4 Image of *Gordonella alkali-eating* under a 40x microscope;

[0046] Figure 5 This is a wavelength scan of lycopene and the sample.

[0047] Figure 6 Linear curves of absorbance values ​​for lycopene reference solutions of different concentrations;

[0048] Figure 7 The effects of seven different feeds on the intestinal morphology of Litopenaeus vannamei;

[0049] Figure 8 The results of the gyrB gene sequence determination for this bacterium;

[0050] Figure 9 The results are the 16S rRNA sequence determination results of this bacterium. Detailed Implementation

[0051] The inventors of this patent isolated a strain of *Gordonia alkanivorans* Weicao-1 from the air in Wuchang. This strain of *Gordonia alkanivorans* Weicao-1 is deposited at the China General Microbiological Culture Collection Center, CGMCC No. 26361.

[0052] Appearance, cell morphology and physiological and biochemical characteristics of *Gordonella alkali-eating*

[0053] like Figure 1-4As shown, when observed on solid culture medium, the colonies of the strain are orange-red to red, round, opaque, with regular edges, a raised center, a smooth surface, and do not spread. Gram staining is positive. Under natural light, the product solution appears as a turbid orange-red to red liquid with precipitate. Under a microscope, the bacteria are short rod-shaped and lack flagella.

[0054] The bacteria used in this product have the ability to produce carotenoids and possess stable genetics and wide adaptability. Even when stored on slant culture media for more than six months and the medium is relatively dry, they can still produce carotenoids and other nutrients. The strain grows rapidly, is highly stable, and contains high yields of carotenoids (calculated as lycopene) and is also rich in peptidoglycan. It can effectively regulate the balance of intestinal flora in animals, regulate the body's immunity, enhance disease resistance, promote health, and improve production performance. Furthermore, testing and analysis have confirmed that this strain is a non-toxic strain that does not produce toxins.

[0055] Table 1 Cell morphology and physiological and biochemical indicators

[0056]

[0057]

[0058] Figure 8 This is the result of the gyrB gene sequence determination of this bacterium. Figure 9 The results are the 16S rRNA sequence determination results of this bacterium.

[0059] Based on a comprehensive analysis of data including similarity in homology, bacterial cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, and gyrB gene sequence, and with reference to relevant papers in Bergey's Manual of Systematic Bacteriology and the International Journal of Systematic and Evolutionary Microbiology, the following identification was made: The bacterium was identified as *Gordonella alkali-eating*.

[0060] Biomass determination of *Gordonia alkanivorans* weicao-1: Plate count method. Results are shown in Table 2.

[0061] Method for detecting carotenoids in oral preparations of *Gordonia alkanivorans* weicao-1:

[0062] Wavelength determination: The test solution and reference solution were prepared according to the methods used in the detection of carotenoid (calculated as lycopene) content. Both the test solution and reference solution were subjected to UV-Vis spectral scanning, with a scanning range of 190-700 nm and a maximum absorbance between 0.1 and 1.5 nm.

[0063] The results showed that the peak shape in the spectrum exhibited typical characteristics of carotenoids, with three absorption peaks at 446 nm, 474 nm, and 504 nm, respectively. Dissolving lycopene standard in acetone and scanning the standard solution revealed a maximum absorption peak at 474 nm, which is consistent with the peak shape and wavelength corresponding to the maximum absorption peak of the extracted sample pigment. (See...) Figure 5 .

[0064] Carotenoid (calculated as lycopene) content detection

[0065] Linearity test

[0066] Accurately weigh lycopene reference standard and prepare solutions containing 0.0, 5.0, 10.0, 15.0, and 20.0 μg / mL. Using acetone as a blank, colorimetric analysis was performed. The absorbance of different concentrations of lycopene reference standard solutions was measured at a wavelength of 474 ± ​​1 nm. A linear curve was plotted between the absorbance of the standard solution and the concentration. The regression equation for the standard curve is: y = 0.0501x + 0.0132, with a correlation coefficient R0. 2 =1, the sample showed good linearity in the range of 0-20.0 μg / mL, see [reference needed]. Figure 6 .

[0067] Preparation of lycopene reference solution (20 μg / mL): Accurately weigh 10 mg (0.01 mg) of lycopene reference standard, dissolve it in acetone to prepare a reference solution containing 20 μg of lycopene per 1 mL.

[0068] Preparation of test solution: Take 5 ml of sample, centrifuge in a high-speed centrifuge for 10 min, discard the supernatant, wash with ultrapure water, centrifuge again, and discard the supernatant. Add 3 ml of 1 mol / L hydrochloric acid (V 盐酸 :V 菌液 Mix the contents of the mixture with a 3:5 ratio, sonicate for 10 minutes, heat in a 100°C water bath for 2 minutes, rapidly cool to below 10°C, centrifuge at high speed, discard the supernatant, wash twice with ultrapure water to obtain cell debris. Then add 5 ml of acetone (V... 丙酮 :V 菌液=1:1), sonicate for 10 min, centrifuge at high speed for 10 min, take the supernatant, repeat this step 3 times, combine the supernatants, put them into a 25 ml volumetric flask, and dilute to volume with acetone; take 2 ml of the solution from the 25 ml volumetric flask, put it into a 10 ml volumetric flask, dilute to the mark with acetone, shake well, and this is the test solution.

[0069] Determination of carotenoid content in samples: Using acetone as a blank, the test solution was measured at a wavelength of 474±1nm.

[0070] Calculation: Carotenoid concentration in the sample (μg / mL): x = (y - 0.0132) × dilution factor / 0.0501

[0071] The results are shown in Table 2.

[0072] Table 2 Sample test results

[0073]

[0074] Regarding the preparation process of oral formulations of *Gordonella alkali-eating*:

[0075] Example:

[0076] When the seeds of *Gordonia alkanivorans* weicao-1 were inoculated onto slant culture medium and cultured at 30-31℃ for 30 hours, they turned red, and microscopic examination showed no contamination and vigorous growth.

[0077] Shake-flask seed culture: Inoculate the slant seed of *Gordonia alkanivorans* weicao-1 into an Erlenmeyer flask at 30-31℃, with a light intensity of 750-850 lx and a shaking speed of 150 rpm for 48 hours. The seed will turn red, and microscopic examination will show no contamination and the seed will be vigorous. Transfer the seed to an inoculation bottle.

[0078] Seed culture: Transfer the seeds from the inoculation bottle into the seed culture tank. The initial pH of the culture medium is 6.8-7.0, the light intensity is 850-1000 lx, the aeration rate is 1:0.9, the defoamer is added automatically, and the temperature is 30-32℃. After 26 hours of culture, the cells turn red and samples are taken for microscopic examination. The cells grow uniformly and are free of contaminants.

[0079] Fermentation tank culture: Inoculate the seed culture from the seed tank into the fermenter at an inoculum rate of 4-8%. Maintain a temperature of 30-32℃, a light intensity of 1000-1500 lx, an aeration rate of 1:1, and automatic addition of defoamer. Culture for 48-52 hours until the culture turns red or orange-red. Microscopic examination reveals that the cells have aged into short rod-like shapes, and the biomass reaches (5-10) × 10⁻⁶. 9Fermentation was stopped when the concentration of carotenoids (calculated as lycopene) was not less than 350 μg / mL. A liquid preparation of *Gordonia alkanivorans* weicao-1 was obtained.

[0080] Culture medium preparation: Prepare the ingredients according to the following formula.

[0081] .

[0082] Oral solid dosage form preparation

[0083] Membrane separation: Using organic membrane equipment, fermentation broth A is subjected to membrane separation and concentrated to one-tenth.

[0084] Refrigerated high-speed centrifugation: Place the above concentrated solution in a refrigerated high-speed centrifuge, centrifuge at 5500-7500 rpm for 10-15 minutes at a temperature below 8°C. Separate the solid and liquid components, and keep the wet bacterial cells and supernatant for later use.

[0085] Microbial granule preparation: Weigh the wet microbial cells, add 4 times the amount of skim milk powder, mix well, granulate through a 24-mesh sieve, and bake at 60°C or below with a vacuum of -0.08 MPa for 15-20 minutes.

[0086] Granulation of the clear liquid: Take the clear liquid and concentrate it to one-eighth of its volume, weigh it, add three times the amount of maltodextrin to refine the soft material, granulate it through a 24-mesh sieve, and bake it at 70-75°C under a vacuum of -0.07-0.08 MPa for 2-3 hours.

[0087] Mixing: Mix the granules from the above steps to obtain the *Gordonia alkanivorans* weicao-1 solid granule formulation, with a biomass of (1.5–2.5) × 10⁻⁶. 10 cfu / g.

[0088] Five batches were produced according to the above process and tested according to the quality control items and testing methods in Example 2.

[0089] Effects of Gordonella alkali-eating bacteria weicao-1 preparation on the diet of weaned piglets

[0090] Effects of *Gordonella alkali* weicao-1 preparation on growth performance, serum biochemistry, antioxidant and immune indicators in weaned piglets

[0091] Experimental Design

[0092] The experiment employed a single-factor experimental design, selecting 144 healthy Duroc-Landrace-Large White crossbred piglets at 28 days of age with an initial weight of approximately 8.65 kg. These piglets were randomly divided into four groups: A (control group), and B, C, and D (experimental groups), with six replicates per group and six piglets per replicate. (Control group A received the basal diet, while groups B, C, and D received supplemental diets supplemented with 0.1%, 0.2%, and 0.4% of *Gordonella alkaliflorus* weicao-1, respectively.) The detailed experimental design is shown in Table 1, and the experiment lasted for 30 days.

[0093] Table 3 Experimental Design

[0094]

[0095] Measurement indicators:

[0096] Growth performance: ADG / (kg / d), ADFI / (kg / d), F / G

[0097] Serum biochemistry: A fully automated biochemical analyzer measures routine serum biochemical indicators.

[0098] Antioxidant: Nanjing Jiancheng kit was used to determine serum glutathione, SOD activity and malondialdehyde (MDA) content, etc.

[0099] Immune indicators: IgG, IgM, NO, IL-2, IL-12, TNF-α, IFN-γ, and swine fever antibody blocking rate.

[0100] Statistical analysis of data: Raw data were initially processed using Excel. Analysis of variance was performed using the One-Way ANOVA procedure in SPSS 13.5 software, and multiple comparisons were conducted using Duncan's method. All experimental data are expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant.

[0101] result

[0102] 1. Effects of *Gordonella alkaligrass* weicao-1 preparation on the growth performance of weaned piglets.

[0103] As shown in Table 4, compared with the control group, the feed conversion ratio of experimental group C was significantly lower than that of the control group (P < 0.05).

[0104] Table 4. Effects of *Gordonella alkaligrass* weicao-1 preparation on growth performance of weaned piglets.

[0105]

[0106] Note: If the lowercase letters in the same column heading are the same or there are no lowercase letters, the difference is not significant (P>0.05); if the lowercase letters are completely different, the difference is significant (P<0.05). The same applies to the following table.

[0107] Effects of alkali-eating bacterium Gordons weicao-1 preparation on serum biochemical indicators of weaned piglets

[0108] The serum biochemical parameters of each experimental group are shown in Table 5. With the increase of the amount of *Gordonella alkali-eating* added to the diet, the concentration of UREA in the serum tended to increase. The concentration of UREA in each experimental group was significantly different from that in the control group (P<0.01). There were no significant differences in other blood biochemical parameters compared with the control group (P>0.05).

[0109] Table 5. Effects of *Gordonella alkaligrass* weicao-1 preparation on serum biochemical indicators of weaned piglets.

[0110]

[0111] Effects of alkali-eating bacterium Gordons weicao-1 preparation on serum antioxidant indicators in weaned piglets

[0112] Table 6 shows that with the increase of the amount of *Gordonella alkali* additive in the diet, the total antioxidant capacity of weaned piglets improved. The serum malondialdehyde (MDA) content in experimental groups B and C was significantly lower than that in the control group (P<0.05). Compared with the control group, the levels of superoxide dismutase (SOD) and catalase (CAT) in experimental group C were significantly increased (P<0.05). The activity of glutathione peroxidase (GSH-Px) continuously increased with the increase of *Gordonella alkali* additive, and the activity in experimental groups C and D was significantly higher than that in the control group (P<0.01).

[0113] Table 6. Effects of *Gordonella alkaligrass* weicao-1 preparation on serum antioxidant indicators in weaned piglets.

[0114]

[0115] 4. Effects of *Gordonella alkaligra* weicao-1 preparation on serum immune indicators in weaned piglets

[0116] As shown in Table 7, with the increase of the amount of *Gordonella alkali* additive, there was no significant difference in the blocking rate of IgG, IgM, and classical swine fever antibodies between the experimental groups and the control group (P>0.05). However, the concentration of NO in the serum of experimental groups C and D was significantly higher than that in the control group (P<0.05). Compared with the control group, the levels of IL-2, IL-12, and TNF-α in the serum of experimental groups C and D were significantly increased (P<0.05). Compared with the control group, there was no significant increase in the concentration of IFN-γ in the serum of the experimental groups (P>0.05), but compared with experimental group B, the concentration of IFN-γ in the serum of experimental group C was significantly increased (P<0.05).

[0117] Table 7. Effects of *Gordonella alkaligrass* weicao-1 preparation on serum immune indicators in weaned piglets.

[0118]

[0119] 5. Effects of *Gordonella alkaligra* weicao-1 preparation on intestinal mucosal immune markers in weaned piglets.

[0120] Table 8 shows that the addition of *Gordonella alkaliphila* weicao-1 preparation to the diet had no significant effect on the secretion of SIgA in the intestinal mucosa. However, the concentration of SIgA in each intestinal segment of experimental group C was relatively high. Compared with the control group, the concentration of IL-12 in the jejunum of the experimental group was lower, with the largest decrease in experimental group C (P<0.05). The concentration of IL-12 in the ileum of experimental group C was significantly lower than that of the control group (P<0.01).

[0121] Table 8. Effects of *Gordonella alkali-eating* weicao-1 preparation on intestinal mucosal immune indicators in weaned piglets.

[0122]

[0123] As shown in Table 9, the addition of alkali-eating Gordon's bacterium weicao-1 to the diet has a significant inhibitory effect on intestinal Escherichia coli; alkali-eating Gordon's bacterium weicao-1 promotes the growth and colonization of Lactobacillus.

[0124] Table 9. Effects of *Gordonella alkaligra* weicao-1 preparation on the intestinal flora of weaned piglets.

[0125]

[0126] As shown in Table 10, when the diet was supplemented with the alkali-eating bacterium Gordons weicao-1 preparation, in the duodenum, the height of the C villi in the experimental group reached the highest value with the increase of the dose of alkali-eating bacterium Gordons weicao-1, and the overall trend was increasing. The crypt depth in the experimental group was smaller than that in the control group. The number of goblet cells showed an overall increasing trend, and there was no significant difference in the number of lymphocytes among the groups (P>0.05).

[0127] In the jejunum, with the increase of the dose of *Gordonella alkaliphila* weicao-1, the villus height gradually increased, reaching an inflection point in group C, with no significant difference between groups (P>0.05). The crypt depth in experimental group C was significantly different from that in group B (P<0.05). The number of goblet cells generally showed an increasing trend, reaching its maximum in experimental group C, with no significant difference between groups (P>0.05). The number of lymphocytes generally showed an increasing trend, reaching its maximum in experimental group C, with no significant difference between groups (P>0.05).

[0128] In the ileum, there were no significant differences in villus height and goblet cell number among the groups (P>0.05), and no obvious trend of change. The crypt depth showed a decreasing trend, reaching the minimum value in experimental group C, with no significant difference among the groups (P>0.05). The number of lymphocytes showed an overall increasing trend, reaching the maximum value in experimental group C, with no significant difference among the groups (P>0.05).

[0129] Table 10. Effects of *Gordonella alkaliflora* weicao-1 preparation on intestinal morphology of weaned piglets.

[0130]

[0131] in conclusion

[0132] 1. Adding the alkali-eating Gordon's bacterium weicao-1 preparation to the diet of weaned piglets can significantly reduce the feed conversion ratio. The effect is best when the dosage of alkali-eating Gordon's bacterium is 0.2%.

[0133] 2. Adding *Gordonella alkali-fedica* weicao-1 to the diets of weaned piglets significantly reduced serum MDA levels and significantly increased serum GSH-Px levels. CAT and T-AOC levels also showed an increasing trend. Overall, the antioxidant data showed the best effect at a dosage of 0.2%. This indicates that *Gordonella alkali-fedica* can effectively improve the antioxidant capacity of piglets.

[0134] 3. Adding the alkali-eating bacterium Gordons weicao-1 preparation to the diet of weaned piglets significantly increased the levels of NO, IL-2, IL-12 and TNF-α in serum and promoted the secretion of SIgA in the intestinal mucosa, indicating that alkali-eating bacterium Gordons can significantly promote humoral immunity and enhance the disease resistance of weaned piglets.

[0135] 4. Adding *Gordonella alkali-federiae* weicao-1 to weaned piglet diets significantly inhibits the proliferation of *Escherichia coli* and promotes the growth of *Lactobacillus*, effectively regulating the microecological balance and thus reducing the diarrhea rate. Simultaneously, *Gordonella alkali-federiae* weicao-1 promotes healthy intestinal development, especially in the jejunum, increases villus height, reduces crypt depth, effectively promotes the proliferation of goblet cells and lymphocytes in the intestinal epithelium, improves the small intestine's ability to absorb nutrients, and thus enhances production performance.

[0136] Effects of Gordonella alkali-eating strain weicao-1 preparation on fattening pig diets

[0137] Effects of *Gordonella alkali-eating* weicao-1 preparation on growth performance, serum biochemical indicators and meat quality of fattening pigs

[0138] Experimental Design

[0139] The experiment employed a single-factor experimental design, selecting 240 healthy Duroc-Landrace-Large White crossbred piglets with an initial weight of approximately 80.16 kg. These piglets were randomly divided into 5 groups, with 8 replicates per group and 6 piglets per replicate. (Control group A received the basal diet; groups B, C, D, and E were supplemented with 0.1%, 0.2%, 0.4%, and 0.6% of *Gordonella oleracea* weicao-1, respectively.) The detailed experimental design is shown in Table 11.

[0140] Table 11 Experimental Design

[0141]

[0142] Measurement indicators:

[0143] Growth performance: ADG / (kg / d), ADFI / (kg / d), F / G

[0144] Serum biochemical antioxidant and tissue antioxidant: The fully automated biochemical analyzer measured routine biochemical indicators in serum, and Nanjing Jiancheng reagent kits were used to determine glutathione, SOD activity and malondialdehyde (MDA) content in serum and tissue.

[0145] Meat quality: pH, meat color, drip loss, tenderness, muscle fat, amino acids (Germany Sykam fully automated amino acid analyzer).

[0146] Statistical analysis of data: Raw data were initially processed using Excel. Analysis of variance was performed using the One-Way ANOVA procedure in SPSS 13.5 software, and multiple comparisons were conducted using Duncan's method. All experimental data are expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant.

[0147] result

[0148] 1. Effects of *Gordonella alkaliphaga* weicao-1 preparation on the growth performance of finishing pigs.

[0149] As shown in Table 12, compared with the control group, the feed conversion ratio of experimental group D was significantly lower than that of the control group (P < 0.05).

[0150] Table 12 Effects of *Gordonella alkali-eating* weicao-1 preparation on growth performance of finishing pigs

[0151]

[0152] Note: If the lowercase letters on the same side of the data are the same or there are no lowercase letters, the difference is not significant (P>0.05). If the lowercase letters are completely different, the difference is significant (P<0.05).

[0153] 2. Effects of *Gordonella alkaligra* weicao-1 preparation on serum biochemistry in fattening pigs

[0154] The effects of different doses of *Gardenia oryzae* weicao-1 preparation on serum biochemical indicators of fattening pigs are shown in Table 13. The concentrations of total protein (TP), albumin (ALB), high-density lipoprotein cholesterol (HDL-C), and alkaline phosphatase (ALP) in serum all showed an increasing trend, while the concentrations of triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) showed a decreasing trend. This indicates that *Gardenia oryzae* weicao-1 preparation can improve the absorption and metabolism of protein in fattening pigs. There was no significant difference in serum aspartate aminotransferase among the groups (P>0.05), indicating that *Gardenia oryzae* weicao-1 preparation did not damage the liver of the experimental pigs and is completely non-toxic.

[0155] Table 13 Effects of *Gordonella alkali-eating* weicao-1 preparation on serum biochemical indicators of fattening pigs

[0156]

[0157] 3. Effects of *Gordonella alkaligra* weicao-1 preparation on serum and tissue antioxidant activity in fattening pigs.

[0158] The effects of different doses of *Gordonella alkali* weicao-1 preparation on serum antioxidant indicators in fattening pigs are shown in Table 14.

[0159] Table 14 Effects of *Gordonella alkaligrass* weicao-1 preparation on serum antioxidant indicators in fattening pigs

[0160]

[0161] The effects of different doses of *Gordonella alkali* weicao-1 preparation on antioxidant indices of the longissimus dorsi muscle in fattening pigs are shown in Table 15. In terms of SOD activity in the longissimus dorsi muscle tissue, the SOD activity of group D (0.2%) was significantly higher than that of the control group (P < 0.05). In terms of MDA content in the tissue, groups D (0.4%) and E (0.6%) were significantly lower than those of the control group (P < 0.05). In terms of GSH-PX activity in the tissue, both groups were higher than those of the control group.

[0162] Table 15 Effects of *Gordonella alkali-eating* weicao-1 preparation on antioxidant indices of the longissimus dorsi muscle in fattening pigs

[0163]

[0164] 4. Effects of *Gordonella alkali-eating* weicao-1 preparation on the quality of fattening pork.

[0165] The results are shown in Table 16. With the increase of the dosage of the alkali-eating bacterium weicao-1 preparation, the pH change of the head and semispinalis muscle in the experimental group C (0.2%) was relatively small within the same time period.

[0166] Table 16 Effects of *Gordonella alkali-eating* weicao-1 preparation on pH of the semispinalis muscle of fattening pigs

[0167]

[0168] The results are shown in Table 17. With the increase of the dosage of the alkali-eating bacterium weicao-1 preparation, the flesh color L* value increased, and the flesh color L* value of D (0.4%) was the lowest.

[0169] Table 17 Effect of *Gordonella alkali-eating* weicao-1 preparation on L* value of fattening pork.

[0170]

[0171] The results are shown in Table 18. The effect of the alkali-eating bacillus weicao-1 preparation on the color b* value of fattening pork showed that the color b* value increased with time, and the D (0.4%) dose group had the lowest color b* value.

[0172] Table 18 Effects of *Gordonella alkaligrass* weicao-1 preparation on the color b* value of fattening pork.

[0173]

[0174] The effects of the alkali-eating bacterium Gordons weicao-1 preparation on the color a* value of fattening pork are shown in Table 19. With the increase of time, the color a* value of meat increased, and the color a* value of meat in the E (0.6%) dosage group was the largest.

[0175] Table 19 Effects of *Gordonella alkaligrass* weicao-1 preparation on color a* value of fattening pork

[0176]

[0177] The effects of different doses of *Gordonella alkali* weicao-1 preparation on the tenderness, drip loss, and muscle fat content of the longissimus dorsi muscle in fattening pigs are shown in Table 20. The E (0.6%) experimental group showed a 3.20% reduction compared to the control group. Regarding drip loss, the B (0.1%), C (0.2%), and D (0.4%) groups showed reductions of 27.54%, 20.34%, and 38.14% compared to the control group, respectively. Compared to the control group, the C (0.2%) and E (0.6%) experimental groups showed significantly increased intramuscular fat (P < 0.05).

[0178] Table 20 Effects of *Gordonella alkali-eating* weicao-1 preparation on tenderness, drip loss, and muscle fat content of the longissimus dorsi muscle in fattening pigs.

[0179]

[0180] The effects of the *Gordonella alkali-eating* weicao-1 preparation on the fatty acid content of the longissimus dorsi muscle in fattening pigs are shown in Table 21. The results showed that a total of 26 fatty acids were detected, all of the same type, mainly composed of C16 and C18 fatty acids, with oleic acid (C18:1) having the highest content. Ten saturated fatty acids and nine polyunsaturated fatty acids were also detected.

[0181] Table 21 Effects of *Gordonella alkali-eating* weicao-1 preparation on fatty acid content in the longissimus dorsi muscle of fattening pigs

[0182]

[0183] The effects of different doses of *Gordonella alkali* weicao-1 preparation on the amino acid content of fresh longissimus dorsi muscle meat of fattening pigs are shown in Table 22. The total amino acid content of fresh longissimus dorsi muscle meat of fattening pigs in each experimental group was higher than that in the control group, and the E (0.6%) group was significantly higher than that in the control group (P < 0.05). As for essential amino acids, the content of each experimental group was higher than that in the control group, and the C (0.2%) and E (0.6%) groups were significantly higher than that in the control group (P < 0.05). As for flavor amino acids, there was no significant difference in the increasing trend between each experimental group and the control group (P > 0.05).

[0184] Table 22 Effects of *Gordonella alkali-eating* weicao-1 preparation on amino acid content of fresh longissimus dorsi muscle in fattening pigs

[0185]

[0186] The effects of different doses of *Gordonella alkali* weicao-1 preparation on the inosinic acid content of fresh longissimus dorsi muscle in fattening pigs are shown in Table 23. The inosinic acid content of each experimental group showed an increasing trend compared with the control group, but the differences were not significant (P>0.05).

[0187] Table 23 Effects of *Gordonella alkali-eating* weicao-1 preparation on inosine content in the longissimus dorsi muscle of fattening pigs

[0188]

[0189] in conclusion:

[0190] 1. Adding alkali-eating bacteria Weicao-1 to the diet can significantly improve the daily weight gain of fattening pigs and significantly reduce the feed conversion ratio by regulating gastrointestinal enzyme activity.

[0191] 2. The alkali-eating bacterium Gordons weicao-1 preparation significantly or extremely significantly increases the GSH-PX and SOD enzyme activities in the serum and tissues of fattening pigs, reduces the MDA content, decreases the degree of muscle oxidation, and improves meat quality.

[0192] 3. The recommended dose of *Gordonella alkali-eating* weicao-1 in the basal diet of fattening pigs is 0.4%.

[0193] Effects of Gordonella alkali-eating strain weicao-1 in Litopenaeus vannamei diet

[0194] Experimental Design

[0195] Shrimp of uniform size (approximately 1.7g in weight) were selected and randomly divided into groups for the experiment. Each aquarium (500L) contained 30 shrimp, with four replicates per treatment. If shrimp died after 15 days of culture, only their weight was recorded; no replacements were made. During the experiment, shrimp were fed three times daily (morning, noon, and evening). Feed was weighed one hour after each feeding, and the amount fed was recorded. The experiment lasted eight weeks, during which the total weight and number of shrimp in each tank were weighed and recorded. Secretory immunoglobulins, immune performance indicators, enzyme activity, and gut microbiota were measured and analyzed. The effects of different diets on the growth performance, digestive metabolism, and hepatobiliary health of Litopenaeus vannamei were then analyzed.

[0196] Data processing

[0197] Formulas for calculating growth and morphological indicators:

[0198] Initial average weight (IBW) (g) = Total weight of shrimp at the start of the experiment / Total number of shrimp at the start of the experiment

[0199] Final average weight (FBW) (g) = Total weight of shrimp at the end of the experiment / Total number of shrimp at the end of the experiment

[0200] Weight gain rate (WG) (%) = (Final average weight - Initial average weight) / Initial average weight × 100

[0201] Specific growth rate (SGR) (%) = (ln final average weight - ln initial average weight) / number of days in the experiment × 100

[0202] Feed conversion ratio (FCR) = Total feed intake during the experiment (g) / Total weight gain of shrimp at the end of the experiment (g)

[0203] Survival rate SR (%) = (Number of shrimp tails at the end / Number of shrimp tails at the beginning) × 100

[0204] Table 24 Experimental Design

[0205]

[0206] Statistical analysis

[0207] All results in this experiment are expressed as mean ± standard error. SPSS 26.0 statistical software was used for data analysis. One-way ANOVA and Duncan's multiple tests were used to compare the statistical significance of differences between groups (P < 0.05).

[0208] result

[0209] Effects of different feeds on the growth and morphology of Litopenaeus vannamei

[0210] The effects of different feeds on the growth and morphological indicators of Litopenaeus vannamei are shown in Table 25. Compared with group A (20% fishmeal group), group B (10% fishmeal + 10% mealworm meal group) showed significantly lower final weight, weight gain rate, specific growth rate, and feed intake (P < 0.05), indicating that high-proportion fishmeal substitution negatively impacted the growth performance of Litopenaeus vannamei. Compared with group B, group F showed significantly increased final weight, weight gain rate, specific growth rate, and feed intake (P < 0.05), with no statistically significant difference compared to group A (P > 0.05). In conclusion, adding 0.05% *Gordonella alkali* weicao-1 to the diet significantly improved the growth performance of shrimp, and the growth performance of shrimp in a 10% fishmeal + 10% mealworm meal diet could reach the level of a 20% fishmeal diet.

[0211] Table 25 Effects of different feed products on the growth and morphology of Litopenaeus vannamei.

[0212]

[0213] Effects of different feeds on the digestibility of Litopenaeus vannamei

[0214] The results of different feeds on the growth and morphological indicators of Litopenaeus vannamei are shown in Table 26: Compared with group A (20% fishmeal group), the dry matter digestibility of Litopenaeus vannamei in group B (10% fishmeal + 10% yellow mealworm meal group) was significantly reduced (P < 0.05), indicating that the high proportion of fishmeal substitution significantly reduced the dry matter digestibility of Litopenaeus vannamei; Compared with group B, the dry matter digestibility of the experimental groups with added alkali-eating bacteria Weicao-1 preparation was significantly improved (P < 0.05); Compared with group A, there was no statistically significant difference in the dry matter digestibility of Litopenaeus vannamei in groups E and F (P > 0.05). In conclusion, adding 0.04%-0.05% of *Gordonella alkali-eating* weicao-1 preparation to the diet can significantly improve the dry matter digestibility of shrimp feed. Furthermore, when 0.04%-0.05% of *Gordonella alkali-eating* weicao-1 preparation is added to a diet containing 10% fishmeal and 10% yellow mealworm meal, the dry matter digestibility of shrimp can reach 20% of that of fishmeal.

[0215] Table 26 Effects of different feeds on the digestibility of Litopenaeus vannamei

[0216]

[0217] Effects of different feeds on hepatopancreatic digestive enzyme activity in Litopenaeus vannamei

[0218] The results of hepatopancreatic digestive enzyme activities in Litopenaeus vannamei are shown in Table 27: Compared with group A (20% fishmeal group), the hepatopancreatic lipase and amylase activities in group B (10% fishmeal + 10% mealworm meal group) were significantly reduced (P < 0.05), indicating that high-proportion fishmeal substitution impairs the digestive capacity of the hepatopancreas in Litopenaeus vannamei; Compared with group B, the hepatopancreatic lipase activities in groups C, D, E, and F were significantly increased (P < 0.05), and the hepatopancreatic amylase activities in groups C, D, and E were significantly increased (P < 0.05). The hepatopancreatic lipase and amylase activities in group E were higher than those in groups C and D (P < 0.05); Compared with group A, the hepatopancreatic lipase activity in group E was significantly increased (P < 0.05), while there was no statistically significant difference in amylase activity in group E (P > 0.05). In conclusion, adding 0.03%-0.05% of the alkali-eating bacterium Gordons weicao-1 preparation to the diet can significantly improve the lipase and amylase activities of shrimp hepatopancreas. Furthermore, when 0.04% of the alkali-eating bacterium Gordons weicao-1 preparation is added to a diet containing 10% fishmeal and 10% yellow mealworm meal, the digestive enzyme activity of shrimp hepatopancreas can reach the level of 20% of that in fishmeal diets.

[0219] Table 27 Effects of different diets on hepatopancreatic digestive enzyme activity in Litopenaeus vannamei

[0220]

[0221] Effects of different feeds on intestinal morphology

[0222] From the sliced ​​image ( Figure 7 From the perspective of different feeds, no significant histological changes were observed in the hindgut of Litopenaeus vannamei. The intestines of all treatment groups maintained good tissue morphology and no obvious pathological changes, indicating a good farming environment. The effects of different feeds on the intestinal tissue morphology of Litopenaeus vannamei are shown in Table 28: Compared with group A (20% fishmeal group), group B (10% fishmeal + 10% mealworm meal group) showed a significant decrease in intestinal villus height and a significant increase in intestinal wall muscle layer thickness (P < 0.05), indicating that high proportion of fishmeal substitution damaged intestinal health; compared with group B, group E showed a significant increase in intestinal villus height (P < 0.05), and groups E and F showed a significant increase in intestinal villus width (P < 0.05); the intestinal wall muscle layer thickness of the groups treated with the alkali-containing Gordon's bacteria weicao-1 preparation was significantly lower than that of group B (P < 0.05). In conclusion, adding 0.04% of the alkali-eating bacterium Gordons weicao-1 preparation to the diet can significantly improve the intestinal villus height, intestinal villus width, and intestinal wall muscle layer thickness in shrimp, and can improve to or even exceed 20% fishmeal levels.

[0223] Table 28 Effects of different diets on intestinal morphology of Litopenaeus vannamei

[0224]

[0225] Effects of different diets on the relative expression levels of antioxidant genes in the hepatopancreas of Litopenaeus vannamei

[0226] The effects of different feeds on the relative expression of antioxidant genes in the hepatopancreas of Litopenaeus vannamei are shown in Table 29: Compared with group A, the relative expression levels of CAT and GSH-Px genes in group B were significantly decreased (P < 0.05), indicating that high-proportion fishmeal substitution impairs the antioxidant capacity of the hepatopancreas of Litopenaeus vannamei. Compared with group B, the relative expression levels of SOD and HSP-70 in groups E, F, and G were significantly upregulated (P < 0.05), while the relative expression levels of GSH-Px genes in groups E and F and the relative expression levels of CAT genes in groups E and G were significantly increased (P < 0.05). In addition, the relative expression levels of SOD, CAT, GSH-Px, and HSP-70 genes in the hepatopancreas of Litopenaeus vannamei in group E were significantly higher than those in group A. In conclusion, the *Gardenia oryzae* Weicao-1 preparation can mitigate the negative impact of high-proportion fishmeal substitution on the antioxidant function of the hepatopancreas of Litopenaeus vannamei, and an addition of 400 g / T is most suitable.

[0227] Table 29 Effects of different diets on the relative expression levels of antioxidant genes in the hepatopancreas of Litopenaeus vannamei.

[0228]

[0229] Effects of different diets on the relative expression levels of immune-related genes in the hepatopancreas of Litopenaeus vannamei

[0230] The effects of different feeds on the relative expression levels of immune-related genes in the hepatopancreas of Litopenaeus vannamei are shown in Table 30: Compared with group A, the relative expression levels of lzm (lysozyme) and propo (prophenol oxidase) genes in the hepatopancreas of group B showed a decreasing trend, but there was no statistically significant difference (P>0.05); compared with group B, the relative expression level of lzm (lysozyme) gene in groups E, F, and G was significantly increased (P<0.05), and the relative expression level of propo (prophenol oxidase) gene in group F was significantly upregulated (P<0.05). This indicates that the addition of 500 g / T *Weicao-1* can significantly increase the relative expression levels of immune-related genes in the hepatopancreas of Litopenaeus vannamei (P<0.05).

[0231] Table 30 Effects of different diets on the relative expression levels of hepatopancreatic immune-related genes in Litopenaeus vannamei.

[0232]

[0233] Effects of different diets on the relative expression levels of hepatopancreatic inflammation-related genes in Litopenaeus vannamei

[0234] Activation of the TNF-α / NF-κB pathway is associated with inflammation expression. The effects of different diets on the relative expression levels of inflammation-related genes in the hepatopancreas of Litopenaeus vannamei are shown in Table 31: Compared with group A, the relative expression levels of TNF-α and Relish genes in the hepatopancreas were significantly decreased in the groups supplemented with the alkaloid-rich *Gordonella alkali* weicao-1 preparation (P < 0.05), and the relative expression level of the Dorsal gene in the hepatopancreas was significantly downregulated in groups E, F, and G (P < 0.05). This indicates that the alkaloid-rich *Gordonella alkali* weicao-1 preparation has an anti-inflammatory effect.

[0235] Table 31 Effects of different diets on the relative expression levels of hepatopancreatic inflammation-related genes in Litopenaeus vannamei.

[0236]

[0237] in conclusion

[0238] Based on the above experimental results, we can draw the following preliminary conclusions:

[0239] (1) In terms of growth performance, adding 0.05% of the alkali-eating bacterium Gordon's weicao-1 preparation to the diet can significantly improve the growth performance of shrimp;

[0240] (2) Regarding dry matter digestibility, adding 0.04%-0.05% of the alkali-eating bacterium Gordon's weicao-1 preparation to the diet can significantly improve the dry matter digestibility of the feed for shrimp.

[0241] (3) Regarding digestive enzyme activity, the addition of 0.04% *Gordonella alkali* weicao-1 preparation significantly increased the lipase and amylase activity of shrimp hepatopancreas;

[0242] (4) In terms of intestinal morphology, adding 0.04% of the alkali-eating bacterium Gordon's weicao-1 preparation to the diet can significantly improve the height of the intestinal villi, the width of the intestinal villi and the thickness of the intestinal wall muscle layer of shrimp.

[0243] (4) In terms of antioxidant capacity, the alkali-eating bacterium Gordonella Weicao-1 preparation can improve the negative impact of high proportion of fishmeal substitution on the antioxidant function of Litopenaeus vannamei hepatopancreas, and the addition of 400g / T is most suitable.

[0244] (5) In terms of antioxidant capacity, the addition of 500g / T of alkali-eating bacteria (Weicao-1) can significantly increase the relative expression of immune-related genes in the hepatopancreas of Litopenaeus vannamei;

[0245] (6) In terms of inflammatory response, treatment with Alkaloid Gordonella (Weicao-1) generally up- and down-regulates the expression of pro-inflammatory factors and exerts an anti-inflammatory effect.

Claims

1. A species of alkali-eating bacterium, characterized in that: It is *Gordonella alkali-eating* ( Gordonia alkanivorans )weicao-1, its accession number is CGMCC No.26361.

2. The application of *Gordonella alkali-eating* as described in claim 1 in animal feed.

3. A process for preparing an oral formulation of *Gordonella alkali-eating*, characterized in that, The preparation steps are as follows: A. Fermentation: a. Slant seeds: *Gordonella alkali-eating* (… Gordonia alkanivorans Seed culture medium with accession number CGMCC No. 26361 was inoculated onto slant culture medium and incubated at 30-31℃ for 24-36 hours. The culture turned red, and microscopic examination showed no contamination and vigorous growth. b. Shake-flask seed: *Gordonella alkali-eating* ( Gordonia alkanivorans Seed cultured on a slant with accession number CGMCC No. 26361 was inoculated into an Erlenmeyer flask at 30-31℃, with a light intensity of 750-850 lx and a shaking speed of 150 rpm for 48 hours. The culture turned red, and microscopic examination showed no contamination and vigorous growth. The culture was then transferred to an inoculation bottle. c. Seed culture: Transfer the seeds from the inoculation bottle to the seed tank for culture. The initial pH of the culture medium is 6.8-7.0, the light intensity is 850-1000 lx, the aeration rate is 1:0.85-0.9, the antifoaming agent is added automatically, and the temperature is 30-32℃. After culturing for 24-26 hours, the cells turn red and samples are taken for microscopic examination. The cells are uniformly grown and free of contaminants. d. Fermentation tank cultivation: Inoculate the seed culture from the seed tank into the fermenter at an inoculum rate of 4-8%. Maintain a temperature of 30-32℃, a light intensity of 1000-1500 lx, an aeration rate of 1:1, and automatically add antifoaming agent. Cultivate for 48-52 hours until the culture turns red to orange-red. Microscopic examination shows that the cells have aged into short rod-shaped structures, and the biomass reaches (1.0-1.5) × 10⁻⁶. 10 Fermentation was stopped at cfu / ml. The whole-cell solution was required to contain at least 350 μg / ml of carotenoids (lycopene) to obtain *Gordonella alkali-eating*. Gordonia alkanivorans The liquid preparation has the accession number CGMCC No. 26361. B. Formulate into oral solid dosage forms: e. Membrane separation: Using organic membrane equipment, the *Gordonella alkali-eating* obtained in step A is separated. Gordonia alkanivorans The liquid formulation, with accession number CGMCC No. 26361, was subjected to membrane separation and concentrated to one-tenth of its original volume. f. Refrigerated high-speed centrifugation: Place the above concentrated solution in a refrigerated high-speed centrifuge, centrifuge at 5500-7500 rpm, control the temperature below 8℃, and centrifuge for 10-15 minutes; separate the solid and liquid, and reserve the bacterial sludge and supernatant; g. Granulation preparation: Weigh the sludge, add 4 times the amount of skim milk powder, mix well, granulate through a 24-mesh sieve, and bake at below 60℃ and under a vacuum of -0.08Mpa for 15-20 minutes; h. Granulation of the clear liquid: Concentrate the clear liquid to one-tenth of its volume, weigh it, add 3 times the amount of maltodextrin to refine the soft material, granulate it through a 24-mesh sieve, and bake it at 70-75℃ under a vacuum of -0.07-0.08 MPa for 2-3 hours; i. Mixing: Mix the particles from steps g and h above to obtain a biomass of (1.5–2.5) × 10⁻⁶. 10 cfu / g, yielding *Gordonella alkali-eating* ( Gordonia alkanivorans The solid dosage form has the accession number CGMCC No.26361.

4. The preparation process of an oral formulation of *Gordonella alkali-eating* as described in claim 3, characterized in that: For the preparation of the culture medium in steps c and d, the ingredients of the seed tank culture medium, by weight / volume percentage, are: 2.0-2.5g / 100ml sucrose, 2.0-3.0g / 100ml molasses, 0.6-0.7g / 100ml peptone, 0.2-0.4g / 100ml trisodium citrate, 0.06-0.08g / 100ml (NH4)2HPO4, 0.01g / 100ml Na2HPO4, 0.045g / 100ml KH2PO4, 0.01g / 100ml MgSO4, 0.08-0.1g / 100ml yeast extract, with the remainder being water, and a pH of 6.8-7.

2. The fermenter culture medium consists of 2.5-3.0 g / 100 ml sucrose, 3.5-4.0 g / 100 ml molasses, 0.7 g / 100 ml peptone, 0.45-0.5 g / 100 ml trisodium citrate, 0.08-0.10 g / 100 ml (NH4)2HPO4, 0.01 g / 100 ml Na2HPO4, 0.045 g / 100 ml KH2PO4, 0.01 g / 100 ml MgSO4, 0.15-0.17 g / 100 ml yeast extract, with the remainder being water, and a pH of 6.8-7.2.

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